Project Summary: Polycomb Repressive Complex 2 (PRC2) maintains an epigenetic memory of cell identity and controls many fundamental cellular processes, such as stem cell pluripotency maintenance, stem cell differentiation, X- chromosome inactivation, imprinting, and so on. Dysregulation of PRC2 function is associated with a wide spectrum of cancers and developmental disorders. On the molecular level, PRC2 mediates histone H3 lysine 27 trimethylation (H3K27me3), hallmark of gene silencing. Cell type and developmental state-specific PRC2 function largely depends on the dynamic interactome of the complex. PRC2 displays tremendous compositional complexity, which correlates with pleiotropic roles of PRC2 in cell development. The core PRC2 complex consists of EZH1/2, EED, SUZ12 and RBBP4/7. The mammalian paralogs EZH1 and EZH2 are the catalytic subunit of PRC2. The enzymatic activity and chromatin targeting of PRC2 are impacted by a diverse array of accessory subunits, including AEBP2, JARID2, PHF1, MTF2, PHF19 and EPOP, which form two classes of mutually exclusive PRC2 holo complexes. Besides these mostly dedicated accessory subunits, PRC2 also dynamically associates with a plethora of other cellular factors to mediate crosstalk with important cell signaling pathways, for example BRCA1 in DNA damage response and repair, DNMTs in DNA methylation and genomic imprinting, and CTCF in genome structure and organization. While biologically and clinically important PRC2 function has been widely appreciated, the underlying molecular mechanisms are largely lacking. Due to their size and complexity, biochemical reconstitution and structural analysis of the molecular assemblies of PRC2 present a formidable challenge; only recently have we and others started to reveal the structural basis of catalysis, chromatin binding and disease mutation of PRC2. The overarching theme of this MIRA award is focused on the structure and function of PRC2 and aims to understand how they are regulated to control cell proliferation and differentiation. The proposed study will close gaps of understanding in the field by addressing the following three specific questions. (1) How the enzymatic activity of PRC2 is regulated in various cellular contexts? (2) How is PRC2 specifically recruited to CpG island chromatin? (3) How are the structural mechanisms of PRC2 connected to gene regulation during some central biological processes, such as stem cell differentiation and epigenetic memory maintenance during cell division?